The hormonal form of vitamin D, calcitriol, acts through the vitamin D receptor (VDR) to regulate calcium homeostasis, cell proliferation, differentiation, and various immune functions. Defects in calcitriol actions thus have a broad spectrum of manifestations ranging from a lack of effects to hyperactivity. Hereditary resistance to calcitriol (HVDRR) usually results from a mutation in the VDR gene and manifests as rickets. We have used skin fibroblasts from subjects with HVDRR to explore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptor activation pathway, including defects in nuclear hormone uptake and in VDR subcellular targeting. We developed new technologies to explore these abnoramlities at the cellular level. First, fully functional GFP chimeras of VDR and RXR were cloned and stably expressed in kidney and osteoblast cells. Then, we developed photostable and biologically active red fluorescing derivatives of vitamin D. These multicolor fluorescing reagents allowed real-time simultaneous recordings of calcitriol and VDR import into the nucleus by confocal laser scanning microscopy. Our studies revealed that calcitriol uptake into the cytoplasm is dependent on the presence of an intact VDR, showed that calcitriol promotes VDR translocation from the cytoplasm into the nucleus, and indicated that this receptor translocation is essential for carrying the hormone into the nucleus. During ligand-induced translocation, VDR associates with microtubules and with the centrosome. Using site-direceted mutagenesis, we found that the integrity of the activation function 2 domain of VDR is essential for the hormone-induced translocation of VDR into the nucleus and into the centrosome. Studies with vitamin D analogs revealed a correlation between the centrosomal targeting of VDR and the antiproliferative effects of calcitriol analogues. In addition, our studies showed an increased proteasomal degradation of RXR in rat osteosarcoma cells (ROS) and the stable expression of YFP-RXR in ROS cells restored the responsiveness to the antiproliferative effects of calcitriol and retinoids. These studies indicate that downregulation of RXR expression can also cause calcitriol resistance in cancer cells.Another collaborative study with Dr. G.D. Roodman's laboratory (Univ Texas Health Sci Center, San Antonio, TX) explored the mechanisms causing calcitriol hypersensitivity of osteoclast precursors from patients with Paget's disease. Binding studies with our green fluorescent tagged calcitriol revealed an increased VDR affinity for calcitriol in osteoclasts from Paget's patients, which may be responsible for the enhanced calcitriol sensitivity in Paget's disease. We will continue to use GFP chimeras of wild-type and mutant VDR and intracellular vitamin D binding proteins to gain further understanding of the disorders of the vitamin D endocrine system.